This invention relates to the interpretation of data from an assay of a biological cell sample, and more particularly, to quantitative DNA measurements in a tissue section.
Cancer diagnosis and prognosis is largely dependent on the pathologic examination of tissue surgically removed from a patient. The specific diagnosis is made by a pathologist, who classifies the tumor by site and by cell of origin after examining stained histologic sections of the fixed, paraffin-embedded cancer tissue. The prognosis depends on many factors, including the specific diagnosis, the presence and pattern of tumor metastasis, the extent of tumor at its site of origin and its proximity to vital structures, and the tumor grade as assessed by a pathologist. In some organs, such as the prostate, the usual determinants of prognosis are inadequate to provide a patient-specific prognosis, especially when such is desired prior to definitive therapy. Consequently, other prognostic indicators have been sought.
One prognostic indicator which has been valuable in the cancers of certain organs is DNA ploidy, which is the ratio of the quantity of DNA in a cancer cell to that in a normal cell in the resting phase of its growth cycle. In general, tumors with normal resting-phase cellular DNA content (diploid) have a better prognosis than those with twice that amount (tetraploid), and these in turn have a better prognosis than those with abnormal DNA content which is not tetraploid (non-tetraploid aneuploid).
The cellular DNA is located in the nucleus. Various methods have been developed for measuring the DNA content of whole nuclei. These methods do not make it possible for the measured cells to be correlated with their position or appearance in a standard histologic section. Thus, it is likely that normal cells will be measured together with tumor cells. Also, distinct areas of tumor cannot be measured separately. An even more important consideration is that very small samples, such as prostate thin core biopsies, are unsuitable.
All of these limitations have been overcome by measuring the DNA content of nuclei and partial nuclei in Feulgen-stained standard histologic sections. A new problem is created, however, by the inevitable inclusion of partial nuclei among the analyzed nuclei. In many sections, because the nuclear diameter exceeds the section thickness, all of the nuclei in the section will be truncated, resulting in a measured DNA content which is less than that of the whole nuclei which existed prior to sectioning the tissue. One consequence is that some nuclei which should be considered tetraploid are assigned a DNA content corresponding to aneuploid or S-phase nuclei, making the prognosis seem worse than it is. Another consequence is a marked inability to discriminate subpopulations of different ploidy in a mixed sample, making such a case uninterpretable.
Several algorithms to correct for the effect of truncated nuclei in a tissue section have been proposed, all based on spherical model nuclei, and described in various articles in the scientific literature (A Method to Estimate the DNA Content of Whole Nuclei from Measurements Made on Thin Tissue Sections, published in Cytometry, Volume 6, pp. 234-237, in 1985 by M. Bins and F. Takens; Feasibility and Limitations of a Cytometric DNA Ploidy Analysis Procedure in Tissue Sections, published in Zentralblatt Pathologie, Volume 139, pp. 407-417, in 1993 by G. Haroske, et. al.; An Analysis of DNA Cytomicrophotometry on Tissue Sections in a Rat Liver Model, published in Analytic and Quantitative Cytology, Volume 5, pp. 117-123, in 1983 by R. W. McCready and J. M. Papadimitriou). These algorithms have been studied using data from a computer model of tissue sectioning (Possibility of Correcting Image Cytometric Nuclear DNA (Ploidy) Measurements in Tissue Sections: Insights from Computed Corpuscle Sectioning and the Reference Curve Method, published in Analytical and Quantitative Cytology and Histology, Volume 19, pp. 376-386, in 1997 by J. A. Freed; U.S. Pat. No. 5,918,038 to Freed for General Method for Determining the Volume and Profile Area of a Sectioned Corpuscle). All methods perform poorly when the nuclei are non-spherical, except when extraordinary care is taken regarding selection of section thickness and of nuclear profiles for analysis. Also, when the non-spherical nuclei have inhomogeneous intranuclear DNA distribution or when the tissue sections are wavy, the performance of these algorithms is markedly degraded, particularly when the DNA concentration varies from one nucleus to another; generally, such cases are uninterpretable. The reference curve method (RCM), described in Possibility of Correcting Image Cytometric Nuclear DNA (Ploidy) Measurements in Tissue Sections: Insights from Computed Corpuscle Sectioning and the Reference Curve Method, published in Analytical and Quantitative Cytology and Histology, Volume 19, pp. 376-386, in 1997 by J. A. Freed; and in patent application Ser. No. 08/929,273 (allowed) to Freed for Method for Correction of Quantitative DNA Measurements in a Tissue Section, has theoretical and practical advantages over the algorithm of McCready and Papadimitriou (as shown in Conceptual Comparison of Two Computer Models of Corpuscle Sectioning and of Two Algorithms for Correction of Ploidy Measurements in Tissue Sections, published in Analytical and Quantitative Cytology and Histology, Volume 22, pp. 17-25, in 2000 by J. A. Freed). However, the problem of distinguishing subpopulations of different ploidy in a tissue section, which is a precondition for successful analysis, has not been solved in many cases.
In U.S. Pat. No. 5,235,522 to Bacus for Method and Apparatus for Automated Analysis of Biological Specimens and in A Method of Correcting DNA Ploidy Measurements in Tissue Sections, published in Modern Pathology, Vol. 7, pp. 652-664, 1994, an apparatus and method for measuring the DNA content of nuclei in tissue sections is described, as well as a method for correction of DNA measurements necessitated by nuclear truncation. The limitations of this method have been discussed in detail in patent application Ser. Pat. 08/929,073 (allowed) to Freed for Method for Correction of Quantitative DNA Measurements in a Tissue Section. Based as it is on the algorithm of McCready and Papadimitriou, the Bacus method shares the weaknesses of that algorithm. The novel feature is that the Bacus method allows the operator to define classes of attributes, thereby excluding many unwanted truncated nuclei from analysis; such an approach (which they call morphometric filtering) is helpful but may be of limited value because the a priori classes may not accord with the natural classes in the specimen being analyzed. Nuclear profile shape is one of the morphometric filters mentioned in passing in A Method of Correcting DNA Ploidy Measurements in Tissue Sections, published in Modern Pathology, Vol. 7, pp. 652-664, 1994, but how it is obtained and its specific use are not described at all. In U.S. Pat. No. 5,235,522 to Bacus for Method and Apparatus for Automated Analysis of Biological Specimens, perimeter is used to determine a xe2x80x98shape factorxe2x80x99 as one parameter used to define the classes to which a cell object can be assigned, but the specific use of this shape factor is not elucidated. Excepting Conceptual Comparison of Two Computer Models of Corpuscle Sectioning and of Two Algorithms for Correction of Ploidy Measurements in Tissue Sections, published in Analytical and Quantitative Cytology and Histology, Volume 22, pp. 17-25, in 2000 by J. A. Freed, and Improved Correction of Quantitative Nuclear DNA (Ploidy) Measurements in Tissue Sections, published in Analytical and Quantitative Cytology and Histology, Volume 21, pp. 103-112, in 1999 by J. A. Freed, there is no mention at all in any prior art of the specific importance and use of perimeter data, that it can improve the accuracy of ploidy analysis, or that it can improve discrimination of ploidy subpopulations in a mixed population.
Several objects and advantages of the present invention are:
(a) to improve, in a tissue section, discrimination of different ploidy subpopulations in a mixed population;
(b) to increase the proportion of tissue sections in which ploidy analysis can be interpreted correctly or interpreted at all;
(c) to improve, in a tissue section, the accuracy of the determination of quantitative nuclear DNA content of each ploidy subpopulation;
(d) to provide a clear indication of nuclear shape;
(e) to provide a method to more accurately classify tumors into the correct prognostic categories;
(f) to provide a method in which non-spherical nuclei can be more readily analyzed;
(g) to provide a method which exceeds the performance of existing methods; and
(h) to overcome problems created when the internal diploid control nuclei have different characteristics than the nuclei being analyzed.